Combinations of a pyrimidine containing nnrti with rt inhibitors

ABSTRACT

The present invention concerns combinations of a pyrimidine containing NNRTI with nucleoside reverse transcriptase inhibitors and nucleotide reverse transcriptase inhibitors useful for the treatment of HIV infected patients or for the prevention of HIV transmission or infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/360,693, filed Mar. 21, 2019, which is a continuation of U.S.application Ser. No. 15/950,548, filed Apr. 11, 2018, now abandoned,which is a continuation of U.S. application Ser. No. 15/383,076, filedDec. 19, 2016, now abandoned, which is a continuation of U.S.application Ser. No. 14/744,501, filed Jun. 19, 2015, now abandoned,which is a continuation of U.S. application Ser. No. 14/454,045, filedAug. 7, 2014, now abandoned, which is a continuation of U.S. applicationSer. No. 12/574,881, filed Oct. 7, 2009, now U.S. Pat. No. 8,841,310,which is a continuation of U.S. application Ser. No. 10/570,228, filedFeb. 28, 2006, now abandoned, which is the National Stage Entry ofInternational Application No. PCT/EP2004/052028, filed Sep. 3, 2004,which claims the benefit of European Application Nos. 03103668.4 filedOct. 2, 2003, 03103335.0, filed Sep. 10, 2003, 03103319.4, filed Sep. 8,2003, and 03103275.8, filed Sep. 3, 2003 and U.S. ProvisionalApplication Nos. 60/508,486, filed Oct. 3, 2003 and 60/499,771, filedSep. 3, 2003.

FIELD OF THE INVENTION

The present invention concerns combinations of a pyrimidine containingNNRTI with nucleoside reverse transcriptase inhibitors and/or nucleotidereverse transcriptase inhibitors useful for the treatment of HIVinfected patients or for the prevention of HIV transmission orinfection.

BACKGROUND OF THE INVENTION

Despite the fact that significant progress has been made by theintroduction of HAART therapy (Highly Active Anti-Retroviral Therapy),resistance of the HIV virus against nucleoside reverse transcriptaseinhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors(NNRTIs), nucleotide reverse transcriptase inhibitors (NtRTIs), proteaseinhibitors and even the more recent fusion inhibitors is still a majorcause of therapy failure. For instance, half of the patients receivinganti-HIV combination therapy do not respond fully to the treatment,mainly because of resistance of the virus to one or more drugs used.Moreover, it has been shown that resistant virus is carried over tonewly infected individuals, resulting in severely limited therapyoptions for these drug-naive patients. On the International AIDSConference in Paris in July 2003, researchers released that the biggeststudy so far of resistance to AIDS drugs finds that about 10 percent ofall newly infected people in Europe have drug-resistant strains. Smallertests to determine the spread of resistance have been done in thehigh-risk city center of San Francisco. This test showed the highestlevel of resistance at 27 percent.

The pharmacokinetic profile of many commercially availableantiretrovirals does not allow relatively low therapeutic doses. Poorpharmacokinetic profiles often in combination with poor solubilityproperties of the antiretrovirals cause the AIDS patient to face a highpill burden which is particularly undesirable for drug-naïve patients orfirst line therapy. Moreover, as a consequence of the AIDS virus evenresisting antiretroviral combination therapy, a physician will boost theplasma levels of the active drugs in order for said antiretrovirals toregain effectivity against the mutated HIV viruses, the consequence ofwhich is an even higher increase in pill burden. Boosting plasma levelsmay also lead to an increased risk of non-compliance with the prescribedtherapy and to increased side-effects.

Several attempts have been made to date to design combination regimens.For instance, the combination of lamivudine (a nucleoside RT inhibitoralso named 3TC) at a 150 mg dose and zidovudine (a nucleotide RTinhibitor also named AZT) at a 300 mg dose, formulated in an oral tabletand dosed twice daily, or the combination of abacavir sulfate at a doseequivalent to 300 mg abacavir (a nucleoside RT inhibitor), lamivudine ata 150 mg dose and zidovudine at a 300 mg dose, formulated in an oraltablet and dosed twice daily.

WO 93/23021 describes therapeutic combinations for the treatment ofHIV-infections comprising zidovudine and an agent serving to enhance theantiviral activity against HIV populations otherwise resistant tozidovudine.

WO 96/01110 describes a triple combination of zidovudine, lamivudine andloviride, the latter being a non-nucleoside RT inhibitor of the a-APAclass.

An overview of new antiretroviral drugs is given in ClinicalMicrobiology and Infection 2003, Vol. 9:3, pp. 186-193.

WO 03/016306 specifically discloses more than 250 pyrimidine derivativehaving HIV replication inhibiting properties that act as non-nucleosideRT inhibitors (NNRTIs) having the ability to inhibit the replicationboth wild-type and of mutant strains. One of said NNRTIs is4-[[4-[[4-(2-cyanoethenyl)-2,6-dimethylphenyl]-amino]-2-pyrimidinyl]amino]-benzonitrile(herein referred to as TMC278). WO 03/016306 also discloses the methodsto synthesize these compounds. It further discloses combinations of saidNNRTIs with other antiretrovirals, i.e. suramine, pentamidine,thymopentin, castanospermine, dextran (dextran sulfate), foscamet-sodium(trisodium phosphono formate), zidovudine (3′-azido-3′-deoxythymidine,AZT), didanosine (2′,3′-di-deoxyinosine; ddI), zalcitabine(dideoxycytidine, ddC), lamivudine (2′-3′-dideoxy-3′-thiacytidine, 3TC),stavudine (2′,3′-didehydro-3′-deoxythymidine, d4T), abacavir, nevirapine(11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido-[3,2-b:2′,3′-e][1,4]diazepin-6-one),efavirenz, delavirdine, TMC120, TMC125, tenofovir,(S)-8-chloro-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)imidazo-[4,5,1-jk][1,4]benzodiazepine-2(1H)-thione,α-[(2-nitrophenyl)amino]-2,6-dichloro-benzene-acetamide, RO-5-3335,indinavir, ritonavir, saquinavir, lopinavir (ABT-378), nelfinavir,amprenavir, TMC126, BMS-232632, VX-175, T-20, T-1249, AMD-3100 andhydroxyurea.

Notwithstanding existing combination therapy, there is still a need forimproved antiretroviral therapy, more particularly AIDS therapy. Thisneed is particularly acute for therapy that is effective not only onwild type HIV virus, but also on the increasingly more common resistantHIV viruses. It is thus highly desirable especially for first linetherapy to design a combination regimen with a low pill burden thatlimits or even suppresses the recurrence of drug resistant virus andwhich can be used and remains effective for a long term.

It is an object of the invention to provide combinations of more thanone therapeutically effective antiretroviral drug, which combinationscan be used as first line therapy in drug-naïve patients for a longperiod of time.

It is also an object of the invention to provide combinations of morethan one therapeutically effective antiretroviral drug in which theantiretroviral drugs have a complementary resistance profile thuscreating a high resistance barrier and thus allowing a drug-naïvepatient to take the combinations for a long period of time.

Another object of the invention is to provide combinations of more thanone therapeutically active antiretroviral drug wherein each of theactive antiretroviral drugs of the combinations can be administered oncedaily thus reducing the pill burden for the patient.

A further object of the invention is to provide combinations of morethan one therapeutically active antiretroviral drug wherein each of theactive antiretroviral drugs of the combinations can be co-formulated.

Yet a further object of the invention is to provide combinations of morethat one therapeutically active antiretroviral drug wherein atherapeutically effective amount of each of the active antiretroviraldrugs of the combinations can be co-formulated in one singlepharmaceutical formulation.

Another object of the present invention is to provide combinations ofmore than one active antiretroviral drug which combinations can be usedto prevent HIV transmission or infection in humans.

All references cited herein are incorporated by reference.

SUMMARY OF THE INVENTION

Thus in a first aspect, the present invention provides a combinationcomprising (i) TMC278 or a stereoisomeric form thereof; or apharmaceutically acceptable salt thereof; or a prodrug thereof; and (ii)a nucleoside reverse transcriptase inhibitor and/or a nucleotide reversetranscriptase inhibitor; wherein TMC278 and the nucleotide reversetranscriptase inhibitor and/or the nucleoside reverse transcriptaseinhibitor are therapeutically effective HIV inhibitors at a dose thatcan be administered once daily.

Thus in a second aspect, the present invention provides a combinationcomprising (i) TMC278 or a stereoisomeric form thereof; or apharmaceutically acceptable salt thereof; or a prodrug thereof; and (ii)a nucleoside reverse transcriptase inhibitor; wherein TMC278 and thenucleoside reverse transcriptase inhibitor are therapeutically effectiveHIV inhibitors at a dose that can be administered once daily.

In a third aspect there is provided a combination comprising (i) TMC278or a stereoisomeric form thereof; or a pharmaceutically acceptable saltthereof; or a prodrug thereof; and (ii) a nucleotide reversetranscriptase inhibitor; wherein TMC278 and the nucleotide reversetranscriptase inhibitor are therapeutically effective HIV inhibitors ata dose that can be administered once daily.

In a fourth aspect there is provided a triple combination comprising (i)TMC278 or a stereoisomeric form thereof; or a pharmaceuticallyacceptable salt thereof; or a prodrug thereof; and (ii) a nucleosidereverse transcriptase inhibitor; and (iii) a nucleotide reversetranscriptase inhibitor; wherein TMC278 and the nucleotide reversetranscriptase inhibitor and the nucleoside reverse transcriptaseinhibitor are therapeutically effective HIV inhibitors at a dose thatcan be administered once daily.

In a fifth aspect there is provided a triple combination comprising (i)TMC278 or a stereoisomeric form thereof; or a pharmaceuticallyacceptable salt thereof; or a prodrug thereof; and (ii) a nucleosidereverse transcriptase inhibitor; and (iii) a second nucleoside reversetranscriptase inhibitor different from the nucleoside reversetranscriptase inhibitor of (ii); wherein TMC278 and the first and secondnucleoside reverse transcriptase inhibitors are therapeuticallyeffective HIV inhibitors at a dose that can be administered once daily.

In another aspect there is provided a pharmaceutical formulationcomprising a pharmaceutically acceptable carrier and a combination asspecified herein.

The invention also concerns the use of the combinations specified hereinas HIV inhibitors and the use thereof in the treatment of HIV infectedpatients or in the prevention of HIV transmission or infection.

The invention is based on the finding that TMC278 is a potent reversetranscriptase inhibitor that has an extremely high genetic barrier incombination with a favourable pharmacokinetic profile allowing oncedaily dosing. It was surprising to discover that TMC278 has all theseproperties together. This is unusual because one cannot predict whatmutations will be selected in the HIV-1 genome by a given drug, whetherthe mutated virus will have any chance of survival under the pressure ofthe drug, how much drug is needed to limit or to suppress the recurrenceof such mutated virus, and at what frequency such drug has to be givento maintain suppression of the development of a resistant virus that canbreak through the genetic barrier of the drug.

DETAILED DESCRIPTION OF THE INVENTION

As used herein the term ‘therapeutically effective HIV inhibitors at adose that can be administered once daily’ means that the HIV inhibitorsare suitable for dosing every 24 hours. The ‘term suitable for dosingevery 24 hours’ means that the HIV inhibitors are such that they can beadministered every 24 hours and give effective blood plasmaconcentrations of the active ingredients such that they are effective tosuppress HIV infection over a period of 24 hours. The HIV inhibitors foruse in the invention can be dosed every 24 hours.

TMC278 or4-[[4-[[4-(2-cyanoethenyl)-2,6-dimethylphenyl]-amino]-2-pyrimidinyl]-amino]-benzonitrileis a known NNRTI, which can be prepared as described in WO03/016306.TMC278 can be used in base form or, which is preferred, as a suitablepharmaceutically acceptable salt form, in particular as an acid additionsalt form. The pharmaceutically acceptable addition salts are meant tocomprise the therapeutically active non-toxic salt forms. The acidaddition salt forms can be obtained by treating the base form withappropriate acids as inorganic acids, for example, hydrohalic acids,e.g. hydrochloric, hydrobromic and the like; sulfuric acid; nitric acid;phosphoric acid and the like; or organic acids, for example, acetic,propanoic, hydroxyacetic, 2-hydroxy-propanoic, 2-oxopropanoic, oxalic,malonic, succinic, maleic, fumaric, malic, tartaric,2-hydroxy-1,2,3-propanetricarboxylic, methanesulfonic, ethanesulfonic,benzene-sulfonic, 4-methylbenzenesulfonic, cyclohexanesulfamic,2-hydroxybenzoic, 4-amino-2-hydroxybenzoic and the like acids. Preferredfor use in the present invention are the hydrohalic acid salts, inparticular the hydrochloride salt.

TMC278 occurs in stereoisomeric forms, more in particular as E- andZ-isomeric forms. Both isomers may be used in the combinations of thepresent invention. Whenever reference is made herein to TMC278, the E-and the Z-form as well as any mixture of both forms are meant to beincluded.

A preferred form of TMC278 for use in the invention is the E-isomer,i.e.(E)-4-[[4-[[4-(2-cyanoethenyl)-2,6-dimethylphenyl]-amino]-2-pyrimidinyl]-amino]-benzonitrile(hereinafter called E-TMC278). The Z-isomer of TMC278, i.e.(Z)-4-[[4-[[4-(2-cyanoethenyl)-2,6-dimethylphenyl]-amino]-2-pyrimidinyl]-amino]-benzonitrile(hereinafter called compound Z-TMC278) can also be used. It hasrelatively high potency against wild-type HIV-1 but is less activeagainst single and double mutants in comparison to the E-isomer. Table 1shows the IC₅₀ value in nM of the E and Z-isomer of TMC278.

TABLE 1 HIV RT mutation E-isomer Z-isomer Wild-type 0.4 0.6 100I 0.4 6.3103N 0.3 1.6 181C 1.3 5.0 188L 2.0 32 227C 2.0 4.0 100I + 103N 7.9 790103N + 181C 1.0 40 227L + 106A 1.0 4.0Whenever reference is made herein to the E-form of TMC278 (i.e.E-TMC278), the pure E-isomer or any isomeric mixture of the E- and theZ-forms wherein the E-form is predominantly present is meant to becomprised, i.e. an isomeric mixture containing more than 50% or inparticular more than 80% of the E-form, or even more than 90% of theE-form. Of particular interest is the E-form substantially free of theZ-form. Substantially free in this context refers to E-Z-mixtures withno or almost no Z-form, e.g. isomeric mixtures containing as much as90%, in particular 95% or even 98% or 99% of the E-form. Equally,whenever reference is made herein to the Z-form of TMC278 (i.e.Z-TMC278), the pure Z-isomer or any isomeric mixture of the Z- and theE-forms wherein the Z-form is predominantly present is meant to becomprised, i.e. an isomeric mixture containing more than 50% or inparticular more than 80% of the Z-form, or even more than 90% of theZ-form. Of particular interest is the Z-form substantially free of theE-form. Substantially free in this context refers to E-Z-mixtures withno or almost no E-form, e.g. isomeric mixtures containing as much as90%, in particular 95% or even 98% or 99% of the Z-form.

Also meant to be included for use in this invention are salts of theisomeric forms of TMC278, in particular the salts mentioned above. Ofparticular interest are Z-TMC278 hydrochloride and specifically E-TMC278hydrochloride.

Advantageously, the nucleotide reverse transcriptase inhibitor and thenucleoside reverse transcriptase inhibitor select mutations in thereverse transcriptase that do not cause resistance to TMC278. Ofparticular interest therefore is any combination specified hereinwherein (1) TMC278 and the nucleoside/nucleotide reverse transcriptaseinhibitor inhibitors are therapeutically effective HIV inhibitors at adose that can be administered once daily and (2) thenucleoside/nucleotide reverse transcriptase inhibitor inhibitors selectmutations in the reverse transcriptase that do not cause resistance toTMC278.

Specifically, in one embodiment, a combination is provided comprising(i) TMC278 or its stereoisomeric form or pharmaceutically acceptablesalt or its prodrug, and (ii) a nucleoside reverse transcriptaseinhibitor, wherein (1) TMC278 and the nucleoside reverse transcriptaseinhibitor are therapeutically effective HIV inhibitors at a dose thatcan be administered once daily and (2) the nucleoside reversetranscriptase inhibitor selects mutations in the reverse transcriptasethat do not cause resistance to TMC278. In another embodiment, acombination is provided comprising (i) TMC278 or its stereoisomeric formor pharmaceutically acceptable salt or its prodrug, and (ii) anucleotide reverse transcriptase inhibitor, wherein (1) TMC278 and thenucleotide reverse transcriptase inhibitor are therapeutically effectiveHIV inhibitors at a dose that can be administered once daily and (2) thenucleotide reverse transcriptase inhibitor selects mutations in thereverse transcriptase that do not cause resistance to TMC278.

In a preferred embodiment, a triple combination is provided comprising(i) TMC278 or its stereoisomeric form or pharmaceutically acceptablesalt or its prodrug, and (ii) a nucleoside reverse transcriptaseinhibitor, and (iii) a nucleotide reverse transcriptase inhibitor,wherein (1) TMC278 and the nucleotide reverse transcriptase inhibitorand the nucleoside reverse transcriptase inhibitor are therapeuticallyeffective HIV inhibitors at a dose that can be administered once dailyand (2) the nucleotide reverse transcriptase inhibitor and thenucleoside reverse transcriptase inhibitor select mutations in thereverse transcriptase that do not cause resistance to TMC278.

In another preferred embodiment, a triple combination is providedcomprising (i) TMC278 or its stereoisomeric form or pharmaceuticallyacceptable salt or its prodrug, and (ii) a nucleoside reversetranscriptase inhibitor, and (iii) a second nucleoside reversetranscriptase inhibitor different from the nucleoside reversetranscriptase inhibitor of (ii); wherein (1) TMC278 and the nucleosidereverse transcriptase inhibitors are therapeutically effective HIVinhibitors at a dose that can be administered once daily and (2) thenucleoside reverse transcriptase inhibitors select mutations in thereverse transcriptase that do not cause resistance to TMC278.

Preferred nucleotide reverse transcriptase inhibitors that can be usedin the combinations subject of this invention include tenofovir and itsprodrug tenofovir disoproxil fumarate.

Tenofovir is an adenosine nucleotide analogue currently commerciallyavailable with activity against retroviruses. Tenofovir disoproxilfumarate (tenofovir DF) is a once-daily, orally administered prodrug oftenofovir. For antiviral activity, tenofovir DF needs to be hydrolysedto the ANP analogue and then phosphorylated to the active diphosphatemoiety [Arimilli et al Antiviral Chemistry and Chemotherapy 1997, 8:6(557-564); Fridland et al. Antiviral Research 1997, 34]. After entry into lymphocytes or macrophages, the prodrug is quantitatively convertedto the parent analogue, tenofovir, and phosphorylated to mono- anddiphosphate metabolites. The cellular enzymes that are responsible forphosphorylation of this drug are adenylate kinase and nucleosidediphosphate kinase [Robbins et al. Antimicrobial Agents and Chemotherapy1995, 39:10 (2304-2308); Robbins et al. Antimicrobial Agents andChemotherapy 1998, 42:3 (612-617)]. Unlike other nucleoside analogues,such as zidovudine or stavudine, both of whose phosphorylation is cellcycle-dependent, tenofovir is efficiently phosphorylated in resting aswell as cycling peripheral blood lymphocytes [Robbins et al. 1998].Tenofovir can inhibit HIV-1 replication in different cell types that maytarget HIV, including primary human blood lymphocytes and macrophages[Perno et al. Antiviral Research 1992 (289-304); Perno et al. MolecularPharmacology 1996, 50:2 (359-366)]. The primary target of tenofovirdiphosphate is reverse transcriptase (RT). Tenofovir diphosphate is acompetitive inhibitor for the incorporation of deoxyadenosinetriphosphate into nascent proviral DNA chains. Inhibition of HIV-1 RT bytenofovir diphosphate has an inhibition constant of approximately 0.9μM, and if the analogue is incorporated into the growing viral DNA chainit may terminate further chain elongation. Tenofovir inhibits viral RTmuch more effectively than it inhibits cellular DNA polymerases [Suo etal Journal of Biological Chemistry 1998, 273:42 (2750-2758)]. Theconcentration required to inhibit the replication of various HIV-1strains by 50% (EC50) in lymphocyte and macrophage cell types (MT-2,CEM, ACH8) ranges from 0.2 to 10 μM. The antiviral effect is achieved atnon-toxic doses of tenofovir (selectivity index ranging from 100 to2000). Tenofovir DF is currently available as 300 mg tablets to be takenonce daily.

Viral resistance to tenofovir in vitro emerges slowly. A recombinantvirus expressing the K65R mutation showed a 3-fold decreasedsusceptibility to tenofovir in vitro [Cherrington et al. InterscienceConference on Antimicrobial Agents and Chemotherapy 1997, 37th].Notably, clinical HIV strains expressing the M184V lamivudine-associatedresistance mutation on RT show wild-type or increased susceptibility totenofovir in vitro, independent of changes in Ki for the mutant enzyme[Miller et al. Interscience Conference on Antimicrobial Agents andChemotherapy 1998,]. Long-term treatment (5 to 15 weeks) of newbornrhesus macaques with tenofovir (doses of 30 mg/kg) starting 3 weeksafter inoculation with simian immunodeficiency virus, resulted inemergence of SIV with approximately 5-fold decreased susceptibility totenofovir [Van Rompay et al. Antimicrobial Agents and Chemotherapy 1996,40:11 (2586-2591)]. This low level of resistance was associated with theappearance of the K65R mutation.

In a preferred embodiment, a combination is provided comprising (i)TMC278 or its stereoisomeric form or pharmaceutically acceptable salt orits prodrug, and (ii) tenofovir or its prodrug tenofovir disoproxilfumarate, wherein TMC278 and tenofovir or its prodrug tenofovirdisoproxil fumarate are therapeutically effective HIV inhibitors at adose that can be administered once daily.

In another preferred embodiment, a triple combination is providedcomprising (i) TMC278 or its stereoisomeric form or pharmaceuticallyacceptable salt or its prodrug, and (ii) a nucleoside reversetranscriptase inhibitor, and (iii) tenofovir disoproxil fumarate;wherein TMC278 and the nucleoside reverse transcriptase inhibitor andtenofovir disoproxil fumarate are therapeutically effective HIVinhibitors at a dose that can be administered once daily.

Preferred nucleoside reverse transcriptase inhibitors that can be usedin the combinations of this invention include abacavir or apharmaceutically acceptable salt thereof, emtricitabine, racemic FTC andlamivudine (also named 3TC).

Emtricitabine or (−)-FTC is the left (−) rotatory enantiomeric form ofracemic FTC or(±)-cis-4-amino-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-2(1H)-pyrimidinone(FTC). It is a commercially available nucleoside analogue and exhibitsactivity against HIV-1 [Hoong et al. Journal of Organic Chemistry 1992(5563-5565); Jeong et al Journal of Medicinal Chemistry 1993, 36:2(181-195); Van Roey et al. Antiviral Chemistry and Chemotherapy 1993,4:6 (369-375]. The in vitro anti-HIV-1 activity of (−)-beta-enantiomerof FTC was reported to be 20-fold more than the (+)-beta-enantiomer, andthe (+)-enantiomer was significantly more toxic than the (−)-enantiomerto myeloid progenitor cells [Schinazi et al Antimicrobial Agents andChemotherapy 1992, 36:11 (2423-2431)]. The potential for HIV-1resistance to FTC was evaluated by serial passage of the virus in humanPBMCs and MT-2 cells in the presence of increasing drug concentrations.Highly drug-resistant HIV-1 variants dominated the replicating viruspopulation after two or more cycles of infection. RT derived fromdrug-resistant viral particles was 15- to 50-fold less susceptible tothe 5′-triphosphate of FTC compared with the enzyme from parental drugsusceptible virus. DNA sequence analysis of the RT gene amplified fromresistant viruses consistently identified mutations at codon 184 fromMet (ATG) to Val (GTG or GTA) [Schinazi et al Antimicrobial Agents andChemotherapy 1993, 37:4 (875-881); Tisdale et al Antiviral Research1993, 20:Suppl 1; Smith et al Journal of Virology 1997, 71:3(2357-2362); Harrer et al Journal of Infectious Diseases 1996, 173:2(476-479); Tisdale et al Proceedings of the National Academy of Sciencesof the United States of America 1993, 90:12 (5653-5656)]. Due to thisobserved single mutation in the YMDD of reverse transcriptase in theHIV-infected patients, (−)-FTC is not suitable for monotherapy and needsto be administered in combination with other antiretroviral agents toeffectively treat patients infected with HIV. Emtricitabine is availableas 200 mg capsules to be taken once a day.

Lamivudine has the chemical name (−)-2′,3′-dideoxy-3′-thiacytidine andis described for instance in EP-382 526 as an antiviral nucleosideanalogue. It is also a well established and useful antiretroviral whichis commercially available for instance as 150 mg oral tablets.Lamivudine is also commercially available in combination with zidovudine(300 mg zidovudine/150 mg lamivudine), and in combination withlamivudine and abacavir sulfate (300 mg zidovudine/150 mglamivudine/equivalent of 300 mg abacavir).

Abacavir is a well established and useful antiretroviral which iscommercially available for instance as an oral solution of abacavirsulfate in a strength equivalent to 20 mg abacavir base/ml, or as anoral tablet of abacavir sulfate in a strength equivalent to 300 mgabacavir base. Abacavir sulfate is also commercially available incombination with lamivudine and zidovudine (300 mg zidovudine/150 mglamivudine/equivalent of 300 mg abacavir).

Abacavir is a carbocyclic nucleoside with potent and selective anti-HIVactivity. Abacavir in its optically active form is disclosed in EP-434450. The cis-isomer of abacavir with unspecified absolute stereochemicalconfiguration is described in EP-349 242. Abacavir is one of the mostpotent NRTI developed to date. An average reduction in viral load ofmore than 1.4 log 10 RNA copies/ml is observed after a short course ofabacavir monotherapy. In vitro, resistant virus is not rapidly selectedby abacavir. A significant decrease in susceptibility to abacavir inwild-type or zidovudine-resistant HIV-1 strains was not observed untilafter eight to ten passages in MT-4 cells. A set of resistance mutationsat HIV reverse transcriptase (RT) codons, 65R, 74V, 115F and/or 184V,are selected during in vitro passage with abacavir, and a combination ofthese mutations was required to confer a 10-fold reduction in abacavirsusceptibility in a laboratory strain of HIV. The first mutationdetected upon passage of HIV-1 in an increasing concentration ofabacavir is M184V, which confers only a 3-fold decrease in HIV-1susceptibility. Phenotype resistance to 3TC and/or the presence of the184V mutation does not prevent viral load response to abacavir therapy.Resistance to multiple nucleosides is associated with a decreased orabsent response to abacavir [Kumar et al Antimicrobial Agents andChemotherapy 1999, 43:3 (603-608); Lanier et al International Conferenceon Retroviruses and Opportunistic Infections 1998, 5th: Chicago; postedon 16 Apr. 1999].

In a preferred embodiment, a combination is provided comprising (i)TMC278 or a stereoisomeric form thereof; or a pharmaceuticallyacceptable salt thereof; or a prodrug thereof, and (ii) emtricitabine,wherein TMC278 and emtricitabine are therapeutically effective HIVinhibitors at a dose that can be administered once daily.

In a preferred embodiment, a combination is provided comprising (i)TMC278 or a stereoisomeric form thereof; or a pharmaceuticallyacceptable salt thereof; or a prodrug thereof; and (ii) lamivudine,wherein TMC278 and lamivudine are therapeutically effective HIVinhibitors at a dose that can be administered once daily.

In another preferred embodiment, a combination is provided comprising(i) TMC278 or a stereoisomeric form thereof; or a pharmaceuticallyacceptable salt thereof; or a prodrug thereof; and (ii) abacavir or apharmaceutically acceptable salt thereof, characterized in that, TMC278and abacavir are therapeutically effective HIV inhibitors at a dose thatcan be administered once daily.

In another preferred embodiment, a combination is provided comprising(i) TMC278 or a stereoisomeric form thereof; or a pharmaceuticallyacceptable salt thereof; or a prodrug thereof; and (ii) abacavirsulfate, characterized in that, TMC278 and abacavir sulfate aretherapeutically effective HIV inhibitors at a dose that can beadministered once daily.

In another preferred embodiment, a triple combination is providedcomprising (i) TMC278 or a stereoisomeric form thereof; or apharmaceutically acceptable salt thereof; or a prodrug thereof; and (ii)emtricitabine, and (iii) a nucleotide reverse transcriptase inhibitor,wherein TMC278 and the nucleotide reverse transcriptase inhibitor andemtricitabine are therapeutically effective HIV inhibitors at a dosethat can be administered once daily.

In another preferred embodiment, a triple combination is providedcomprising (i) TMC278 or a stereoisomeric form thereof; or apharmaceutically acceptable salt thereof; or a prodrug thereof; and (ii)lamivudine, and (iii) a nucleotide reverse transcriptase inhibitor,wherein TMC278 and the nucleotide reverse transcriptase inhibitor andlamivudine are therapeutically effective HIV inhibitors at a dose thatcan be administered once daily.

In another preferred embodiment, a triple combination is providedcomprising (i) TMC278 or a stereoisomeric form thereof; or apharmaceutically acceptable salt thereof; or a prodrug thereof; and (ii)abacavir or a pharmaceutically acceptable salt thereof, or preferablyabacavir sulfate, and (iii) a nucleotide reverse transcriptaseinhibitor, wherein TMC278 and the nucleotide reverse transcriptaseinhibitor and abacavir or a pharmaceutically acceptable salt thereof, orpreferably abacavir sulphate, are therapeutically effective HIVinhibitors at a dose that can be administered once daily.

In another preferred embodiment, a triple combination is providedcomprising (i) TMC278 or a stereoisomeric form thereof; or apharmaceutically acceptable salt thereof; or a prodrug thereof; and (ii)emtricitabine, and (iii) tenofovir or its prodrug tenofovir disoproxilfumarate, wherein TMC278 and emtricitabine and tenofovir or its prodrugtenofovir disoproxil fumarate are therapeutically effective HIVinhibitors at a dose that can be administered once daily.

In another preferred embodiment, a triple combination is providedcomprising (i) TMC278 or a stereoisomeric form thereof; or apharmaceutically acceptable salt thereof; or a prodrug thereof; and (ii)lamivudine and (iii) tenofovir or its prodrug tenofovir disoproxilfumarate, wherein TMC278 and lamivudine and tenofovir or its prodrugtenofovir disoproxil fumarate are therapeutically effective HIVinhibitors at a dose that can be administered once daily.

In another preferred embodiment, a triple combination is providedcomprising (i) TMC278 or a stereoisomeric form thereof; or apharmaceutically acceptable salt thereof; or a prodrug thereof; and (ii)abacavir or a pharmaceutically acceptable salt form thereof, preferablyabacavir sulfate; and (iii) tenofovir or its prodrug tenofovirdisoproxil fumarate, wherein TMC278 and abacavir or a pharmaceuticallyacceptable salt form thereof, preferably abacavir sulfate and tenofoviror its prodrug tenofovir disoproxil fumarate are therapeuticallyeffective HIV inhibitors at a dose that can be administered once daily.

The following preferred triple combinations are also included

-   (a) TMC278 or a stereoisomeric form thereof; or a pharmaceutically    acceptable salt thereof; or a prodrug thereof; with emtricitabine    and tenofovir disoproxil fumarate;-   (b) TMC278 or a stereoisomeric form thereof; or a pharmaceutically    acceptable salt thereof; or a prodrug thereof; with lamivudine and    tenofovir disoproxil fumarate.-   (c) TMC278 or a stereoisomeric form thereof; or a pharmaceutically    acceptable salt thereof; or a prodrug thereof; with abacavir sulfate    and tenofovir disoproxil fumarate.-   (d) TMC278 or a stereoisomeric form thereof; or a pharmaceutically    acceptable salt thereof; or a prodrug thereof; with emtricitabine    and lamivudine;-   (e) TMC278 or a stereoisomeric form thereof; or a pharmaceutically    acceptable salt thereof; or a prodrug thereof; emtricitabine and    abacavir or a pharmaceutically acceptable salt thereof, preferably    abacavir sulfate.-   (f) TMC278 or a stereoisomeric form thereof; or a pharmaceutically    acceptable salt thereof; or a prodrug thereof; abacavir or a    pharmaceutically acceptable salt thereof, preferably abacavir    sulfate and lamivudine.

In particular, in each of the combinations (a)-(f) the activeingredients, in particular TMC278, emtricitabine, lamivudine, abacaviror a pharmaceutically acceptable salt form thereof, preferably abacavirsulfate, and tenofovir or its prodrug tenofovir disoproxil fumarate, aretherapeutically effective HIV inhibitors at a dose that can beadministered once daily.

The double combinations of the present invention may contain one or moreadditional active ingredients, which can be agents useful for treatingHIV infected patients or other active agents. The triple combinations ofthe present invention may equally contain one or more additional activeingredients, which can be agents useful for treating HIV infectedpatients or other active agents. Preferably any of these additionalagents are therapeutically effective at a dose that can be administeredonce daily.

The active ingredients of the combinations of the present invention maybe administered simultaneously, concurrently or sequentially.Simultaneous administration may be done by employing a unitarypharmaceutical formulation or separate pharmaceutical formulations. Ingeneral, the combinations may be administered by topical, oral, rectal,intravenous, subcutaneous or intramuscular routes. For first linetherapy of HIV infection, simultaneous administration employing aunitary pharmaceutical formulation is preferred.

Thus, in another aspect there is provided a product containing acombination as specified herein as a combined preparation forsimultaneous, separate or sequential use against HIV infection.

The invention also provides a product containing (i) TMC278 or astereoisomeric form thereof; or a pharmaceutically acceptable saltthereof; or a prodrug thereof; and (ii) a nucleoside reversetranscriptase inhibitor and/or a nucleotide reverse transcriptaseinhibitor; wherein TMC278 and the nucleotide reverse transcriptaseinhibitor and/or the nucleoside reverse transcriptase inhibitor aretherapeutically effective HIV inhibitors at a dose that can beadministered once daily; as a combined preparation for simultaneous,separate or sequential use against HIV infection.

In a further aspect there is provided a product containing (i) TMC278 ora stereoisomeric form thereof; or a pharmaceutically acceptable saltthereof; or a prodrug thereof; and (ii) a nucleoside reversetranscriptase inhibitor; wherein TMC278 and the nucleoside reversetranscriptase inhibitor are therapeutically effective HIV inhibitors ata dose that can be administered once daily; as a combined preparationfor simultaneous, separate or sequential use against HIV infection.

In another aspect there is provided a product containing (i) TMC278 or astereoisomeric form thereof; or a pharmaceutically acceptable saltthereof; or a prodrug thereof; and (ii) a nucleotide reversetranscriptase inhibitor, wherein TMC278 and the nucleotide reversetranscriptase inhibitor are therapeutically effective HIV inhibitors ata dose that can be administered once daily; as a combined preparationfor simultaneous, separate or sequential use against HIV infection.

In another aspect there is provided a product containing (i) TMC278 or astereoisomeric form thereof, or a pharmaceutically acceptable saltthereof; or a prodrug thereof; and (ii) a nucleoside reversetranscriptase inhibitor; and (iii) a nucleotide reverse transcriptaseinhibitor; wherein TMC278 and the nucleotide reverse transcriptaseinhibitor and the nucleoside reverse transcriptase inhibitor aretherapeutically effective HIV inhibitors at a dose that can beadministered once daily; as a combined preparation for simultaneous,separate or sequential use against HIV infection.

In another aspect there is provided a product containing (i) TMC278 or astereoisomeric form thereof; or a pharmaceutically acceptable saltthereof; or a prodrug thereof; and (ii) a nucleoside reversetranscriptase inhibitor; and (iii) a second nucleoside reversetranscriptase inhibitor other than the nucleoside reverse transcriptaseinhibitor of (ii); wherein TMC278 and the nucleoside reversetranscriptase inhibitors are therapeutically effective HIV inhibitors ata dose that can be administered once daily; as a combined preparationfor simultaneous, separate or sequential use against HIV infection.

The active ingredients in the products of the invention are present intherapeutically effective amounts, the latter meaning an amount that issufficient to exert a sufficient HIV inhibitory effect during a certaintime period, i.e. the time period between each intake of theformulations, preferably for about 24 hours.

Particular embodiments are products as specified above containing one ormore of the specific active ingredients mentioned herein such asemtricitabine, racemic FTC, lamivudin, tenofovir and its prodrugtenofovir disoproxil fumarate.

The products as mentioned above may contain separate formulations of theactive ingredients, or two or where applicable more of the activeingredients may be co-formulated.

In still a further aspect the invention provides pharmaceuticalformulations containing a combination as specified herein and a suitablecarrier.

In another aspect there is provided a pharmaceutical formulationcomprising a pharmaceutically acceptable carrier and as activeingredients (i) TMC278 or a stereoisomeric form thereof; or apharmaceutically acceptable salt thereof; or a prodrug thereof; and (ii)a nucleoside reverse transcriptase inhibitor and/or a nucleotide reversetranscriptase inhibitor; wherein TMC278 and the nucleoside reversetranscriptase inhibitor and/or the nucleotide reverse transcriptaseinhibitor are therapeutically effective HIV inhibitors at a dose thatcan be administered once daily.

The invention further provides a pharmaceutical formulation comprising apharmaceutically acceptable carrier and as active ingredients (i) TMC278or a stereoisomeric form thereof; or a pharmaceutically acceptable saltthereof; or a prodrug thereof; and (ii) a nucleoside reversetranscriptase inhibitor; wherein TMC278 and the nucleoside reversetranscriptase inhibitor are therapeutically effective HIV inhibitors ata dose that can be administered once daily.

In still another aspect there is provided a pharmaceutical formulationcomprising a pharmaceutically acceptable carrier and as activeingredients (i) TMC278 or a stereoisomeric form thereof; or apharmaceutically acceptable salt thereof; or a prodrug thereof; and (ii)a nucleotide reverse transcriptase inhibitor, wherein TMC278 and thenucleoside reverse transcriptase inhibitor and the nucleotide reversetranscriptase inhibitor are therapeutically effective HIV inhibitors ata dose that can be administered once daily.

In still another aspect there is provided a pharmaceutical formulationcomprising a pharmaceutically acceptable carrier and as activeingredients (i) TMC278 or a stereoisomeric form thereof; or apharmaceutically acceptable salt thereof; or a prodrug thereof; and (ii)a nucleoside reverse transcriptase inhibitor; and (iii) a nucleotidereverse transcriptase inhibitor; wherein TMC278 and the nucleosidereverse transcriptase inhibitor and the nucleotide reverse transcriptaseinhibitors are therapeutically effective HIV inhibitors at a dose thatcan be administered once daily.

In still another aspect there is provided a pharmaceutical formulationcomprising a pharmaceutically acceptable carrier and as activeingredients (i) TMC278 or a stereoisomeric form thereof; or apharmaceutically acceptable salt thereof; or a prodrug thereof; and (ii)a nucleoside reverse transcriptase inhibitor; and (iii) a secondnucleoside reverse transcriptase inhibitor different from the nucleosidereverse transcriptase inhibitor of (ii); wherein TMC278 and thenucleoside reverse transcriptase inhibitors are therapeuticallyeffective HIV inhibitors at a dose that can be administered once daily.

The active ingredients in the pharmaceutical formulations of theinvention are present in therapeutically effective amounts, the lattermeaning an amount that is sufficient to exert a sufficient HIVinhibitory effect during a certain time period, i.e. the time periodbetween each intake of the formulations, preferably for about 24 hours.

Particular embodiments are pharmaceutical formulations as specifiedabove containing one or more of the specific active ingredientsmentioned herein such as emtricitabine, racemic FTC, lamivudin,tenofovir and its prodrug tenofovir disoproxil fumarate.

The pharmaceutical formulations of the present invention may beformulated into various forms for different types of administration. Toprepare the pharmaceutical formulations of this invention, effectiveamounts of the active ingredients, optionally in addition salt form, iscombined in intimate admixture with a pharmaceutically acceptablecarrier, which carrier may take a wide variety of forms depending on theform of preparation desired for administration. The pharmaceuticalformulations of the invention are preferably formulated in unitarydosage form suitable, particularly, for administration orally, rectally,percutaneously, or by parenteral injection. For example, in preparingthe formulations in oral dosage form, any of the usual pharmaceuticalmedia may be employed such as, for example, water, glycols, oils,alcohols and the like in the case of oral liquid preparations such assuspensions, syrups, elixirs, emulsions and solutions; or solid carrierssuch as starches, sugars, kaolin, diluents, lubricants, binders,disintegrating agents and the like in the case of powders, pills,capsules, and tablets. Because of their ease in administration, tabletsand capsules represent the most advantageous oral dosage unit forms, inwhich case solid pharmaceutical carriers are obviously employed. Forparenteral compositions, the carrier will usually comprise sterilewater, at least in large part, though other ingredients, for example, toaid solubility, may be included. Injectable solutions, for example, maybe prepared in which the carrier comprises saline solution, glucosesolution or a mixture of saline and glucose solution. Injectablesuspensions may also be prepared in which case appropriate liquidcarriers, suspending agents and the like may be employed.

In one aspect of the invention, the present combinations can beformulated in an oral tablet form further comprising pharmaceuticallyacceptable excipients having a weight ranging between 150 mg and 600 mg,suitable ranging between 200 and 400 mg. Convenient oral tablet formscontaining the active ingredients according to the present inventionhave a total nominal weight ranging between 200 mg and 1500 mg, suitablybetween 500 mg and 1250 mg, more suitable between 600 and 1100 mg.

An advantage of the pharmaceutical formulations of the invention residesin the fact that each of the ingredients of the present combinations canbe co-formulated in one pharmaceutical formulation and do not have to beadministered separately. The daily therapeutic antiretroviral amount ofthe ingredients of the present combinations of such co-formulated singlepharmaceutical form preferably is administered in a single unit dosageform but, if desired, also multiple unit dosage forms, such as two,three, four, five or even more unit dosage forms may be administered. Aphysician will be able to determine the exact dosage to be given takinginto account the severity of the patient's condition as well as thepatient's weight, gender and possibly other parameters such asindividual differences in absorption, biodistribution, metabolism andexcretion rates for each drug as well as other factors known to thoseskilled in the art.

This invention also provides a method of treating HIV infected patientssaid method comprising administering a combination as specified herein.

Furthermore there is provided a method of treating HIV infectedpatients, said method comprising administering TMC278 or itsstereoisomeric form or pharmaceutically acceptable salt or its prodrug,in combination with a nucleoside reverse transcriptase inhibitor and/ora nucleotide reverse transcriptase inhibitor, in which method atherapeutically effective amount of TMC278 and the nucleoside reversetranscriptase inhibitor and/or nucleotide reverse transcriptaseinhibitor can be administered once daily.

Furthermore there is provided a method of treating HIV infectedpatients, said method comprising administering TMC278 or itsstereoisomeric form or pharmaceutically acceptable salt or its prodrug,in combination with a nucleoside reverse transcriptase inhibitor, inwhich method a therapeutically effective amount of TMC278 and thenucleoside reverse transcriptase inhibitor can be administered oncedaily.

In a further aspect of this invention concerns a method of treating HIVinfected patients said method comprising administering TMC278 or itsstereoisomeric form or pharmaceutically acceptable salt or its prodrug,and a nucleotide reverse transcriptase inhibitor, in which method atherapeutically effective amount of TMC278 and the nucleotide reversetranscriptase inhibitor can be administered once daily.

Still a further aspect of this invention comprises a method of treatingHIV infected patients said method comprising administering TMC278 or itsstereoisomeric form or pharmaceutically acceptable salt or its prodrug,in combination with a nucleoside reverse transcriptase inhibitor, and anucleotide reverse transcriptase inhibitor, in which method atherapeutically effective amount of TMC278, the nucleotide reversetranscriptase inhibitor and the nucleoside reverse transcriptaseinhibitor can be administered once daily.

Still a further aspect of this invention comprises a method of treatingHIV infected patients said method comprising administering TMC278 or itsstereoisomeric form or pharmaceutically acceptable salt or its prodrug,in combination with a nucleoside reverse transcriptase inhibitor, and asecond nucleoside reverse transcriptase inhibitor different from theformer nucleoside reverse transcriptase inhibitor, in which method atherapeutically effective amount of TMC278, the nucleoside reversetranscriptase inhibitors can be administered once daily.

The active ingredients in the methods of the invention are administeredin therapeutically effective amounts, the latter meaning an amount thatis sufficient to exert a sufficient HIV inhibitory effect during acertain time period, i.e. the time period between each intake of theformulations, preferably for about 24 hours.

Particular embodiments are methods as specified above wherein one ormore of the specific active ingredients mentioned herein such asemtricitabine, racemic FTC, lamivudin, tenofovir and its prodrugtenofovir disoproxil fumarate, are administered.

One embodiment of the present invention relates to the presentcombinations for use as a medicine. Another embodiment relates to thecombinations of the present invention for use in the manufacture of amedicament to treat HIV infected patients.

Of particular interest are any of the combinations as specified herein,or any of the products, pharmaceutical formulations, unit dosage forms,methods and uses being based on said combinations, wherein TMC278 isE-TMC287, or preferably TMC278 hydrochloride salt or more preferablyE-TMC278 hydrochloride salt.

The combinations of this invention are especially useful for thetreatment of AIDS and related clinical conditions such as AIDS relatedcomplex (ARC), progressive generalised lymphadenopathy (PGL) or AIDSrelated neurological conditions such as multiple sclerosis. The presenttriple combination may be particularly useful for the treatment ofdrug-naïve HIV infected patients.

The combinations of the invention are also useful for the prevention ofHIV transmission or infection in humans, in particular sexualtransmission. Thus, the present invention relates to the use ofcombinations according to the present invention for the manufacture of amedicament for the prevention of HIV infection or transmission viasexual intercourse or related intimate contact between partners. Theinvention also relates to a method of preventing HIV infection ortransmission via sexual intercourse or related intimate contact betweenpartners comprising administering to a subject in need thereof aneffective amount of any of the combinations according to the presentinvention.

The respective daily dose for each of the active ingredients of acombination according to the present invention may range between 10 mgand 800 mg, preferably between 50 and 400 mg, more preferably between 50and 300 mg, or between 100 and 300 mg. In particular, the daily dose forTMC278 may range between 10 mg and 500 mg, preferably between 10 and300, more preferably between 50 and 250 mg, still more preferablybetween 50 and 200 mg, e.g. about 100 mg.

The weight ratio of each couple of components of the triple combinationtaken on a daily basis may vary in a range from 1/10 to 10/1. Suitably,the weight ratio of each couple varies between 1/6 and 6/1, moresuitably 1/4 and 4/1, preferably between 1/3 and 3/1, and morepreferably between 1/2 and 2/1.

Table 2 lists some examples of the daily dose for each of the activeingredients in combinations of compound E-TMC278, emtricitabine andtenofovir.

Combination no. E-TMC278 Emtricitabine Tenofovir 1  50 mg 200 mg — 2  50mg — 300 mg 3 100 mg 200 mg — 4 100 mg — 300 mg 5 200 mg 200 mg — 6 200mg — 300 mg 7  50 mg 200 mg 300 mg 8 100 mg 200 mg 300 mg 9 200 mg 200mg 300 mg

Table 3 lists some examples of the daily dose for each of the activeingredients in combinations of TMC278, abacavir and lamivudine whereinthe dose mentioned in the table for abacavir sulfate is the equivalentdose of abacavir base.

Combination no. E-TMC278 Lamivudine Abacavir sulfate 1  50 mg 150 mg 300mg 2 100 mg 150 mg 300 mg 3 200 mg 150 mg 300 mg

Thus, an interesting combination according to the present inventioncomprises compound E-(A) in a daily dose ranging between 10 mg and 500mg, a daily dose of 150 mg lamivudine and a daily dose of an equivalentof 300 mg abacavir base. Suitably, such combination is formulated in asingle pharmaceutical form.

Another interesting combination according to the present inventioncomprises compound E-(A) in a daily dose ranging between 50 mg and 250mg, a daily dose of 150 mg lamivudine and a daily dose of an equivalentof 300 mg abacavir base. Suitably, such combination is formulated in asingle pharmaceutical form.

The present invention also relates to a pharmaceutical composition in aform adapted to be applied to a site where sexual intercourse or relatedintimate contact can take place, such as the genitals, rectum, mouth,hands, lower abdomen, upper thighs, especially the vagina and mouth,comprising a pharmaceutically acceptable carrier and as activeingredients an effective amount of a combination according to thepresent invention. As appropriate special adapted compositions there maybe cited all compositions usually employed for being applied to thevagina, rectum, mouth and skin such as for example gels, jellies,creams, ointments, films, sponges, foams, intravaginal rings, cervicalcaps, suppositories for rectal or vaginal application, vaginal or rectalor buccal tablets, mouthwashes. To prepare such pharmaceuticalcompositions, an effective amount of each of the particular compounds ofthe triple combination as the active ingredients is combined in intimateadmixture with a pharmaceutically acceptable carrier, which carrier maytake a wide variety of forms depending on the form of administration. Inorder to increase the residence time of such pharmaceutical compositionat the site of administration, it may be advantageous to include in thecomposition a bioadhesive, in particular a bioadhesive polymer. Abioadhesive may be defined as a material that adheres to a livebiological surface such as for example a mucus membrane or skin tissue.

Thus, the present invention also relates to a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and as activeingredients an effective amount of each of the compounds of the presenttriple combination characterized in that the pharmaceutical compositionis bioadhesive to the site of application. Preferably, the site ofapplication is the vagina, rectum, mouth or skin, most preferred is thevagina.

Otten R A et al in Journal of Virology (2000), 74(20), 9771-9775 andWitvrouw M et al in Antiviral Research (2000), 46(3), 215-221 disclosethe ability of tenofovir to delay HIV viral breakthrough after high-risksexual exposure.

Pani A et al in Antiviral Chemistry & Chemotherapy (2001), 12(Suppl. 1),51-59 describe the ability of lamivudine to delay viral breakthrough.

The ability of TMC278 to prevent HIV infection via sexual intercourse orrelated intimate contact between partners can be demonstrated in thefollowing test. Immature monocyte derived dendritic cells (immMO-DC)represent a good model for interstitial dendritic cells, which are earlytargets during sexual HIV transmission and important initiators of theimmune response. These immMO-DC were used in “in vitro” models to testthe prevention of HIV infection via sexual intercourse or relatedintimate contact between partners. One such model is described in theexperimental part and indicates that the TMC278 potently inhibits HIVreplication in MO-DC/CD4(+) T cell co-cultures.

EXAMPLES Example 1: Pharmacokinetics of E-TMC278

A double-blind, randomized, placebo-controlled Phase I trial wasdesigned to evaluate safety, tolerability, and ex-vivo pharmacokineticsof single doses of compound E-TMC278 in healthy male volunteers. Oraldoses of 12.5, 25, and 50 mg were formulated in PEG 400 and taken with astandard meal. The pharmacokinetic results are shown in Table 4.

The pharmacokinetic results of another double-blind, randomized,placebo-controlled Phase I study with 4 dosing sessions to evaluate thesafety, tolerability, pharmacokinetics and ex-vivo pharmacodynamics ofsingle 100 mg and 200 mg oral doses of compound E-TMC278 in healthy malesubjects are also reported in Table 4. Randomization was such that foreach session 6 subjects received the same dose of compound E-TMC278 and3 subjects received placebo. There was a time interval of about 14 daysbetween each dosing session

Table 4 shows that high and dose-proportional exposures were obtained.The correlation coefficient for the 5 C_(max) datapoints is 0.9897 andfor the area under the curve values between 0 and 48 hours(AUC_(0-48 hr)) 0.9952. Half-life of plasma concentrations rangedbetween 37 and 39 hours. The compound was well tolerated by thevolunteers. No relevant adverse effects of the drug were noted.

TABLE 4 Parameter 12.5 mg 25 mg 50 mg 100 mg 200 mg C_(max) (ng/ml)  73± 14 149 ± 32  267 ± 27 482 ± 121 807 ± 207 T_(max) (hr) 4.0 ± 0  4.0 ±1.3  4.0 ± 1.3 4.3 ± 0.8 4.3 ± 0.8 AUC_(0-48 hr) (nghr/ml) 1337 ± 3102805 ± 496  5094 ± 509 8162 ± 2251 15592 ± 2746  AUC_(0-∞) (nghr/ml)2210 ± 473 4637 ± 1164  8872 ± 1342 15844 ± 4592  T_(1/2) (hr) 37.1 38.745 ± 9 55 ± 18

Example 2: Virological Profile of Compound E-TMC278

Compound E-TMC278 was tested in a cell-based assay, using natural hostcells of HIV. MT-4 cells (a cell line of human T cells) were infectedwith HIV-1 (wild type or mutants) and exposed to differentconcentrations of antiviral compound in the presence of 10% fetal calfserum. Cytotoxicity was determined in parallel with the antiviralactivity so that the selectivity of the antiviral effect could beassessed. Active compounds have to penetrate the cell membrane in orderto interfere with replication steps inside the cell.

After four days of incubation at 37° C., the viability of the HIV andmock-infected cells was assessed by an automated tetrazolium-basedcolorimetric assay. This method enabled the calculation of both the 50%inhibitory concentration for inhibition of viral cytopathicity (IC50),the IC90, and the 50% cytotoxic concentration (CC50). The ratioCC50/IC50, also called the selectivity index, is an indication of thespecificity of the antiviral effect. Tested HIV strains included: Wildtype (wt) HIV-1; a panel of single and double mutants, obtained bysite-directed mutagenesis (SDM), and a panel of clinical isolates,selected for resistance against NNRTIs.

Activity Towards Wild Type and SDM Mutants

A limited panel of HIV-1 mutants was constructed using site-directedmutagenesis (SDM) and homologous recombination techniques. CompoundE-TMC278 was tested against an extended panel of single and doublemutants known to be resistant against commercially available NNRTIs.Nevirapine (NVP) and efavirenz (EFV) were included as controls.

The results are shown in Table 5 (values presented are IC50 values innM). For wild type virus, the obtained IC50 was 0.4 nM (0.15 ng/ml) andthe IC90 1.3 nM (0.48 ng/ml). The HIV strain with the lowest sensitivityagainst compound E-TMC278 within this selection was the double mutant100I+103N, with an IC50 of about 8 nM and an IC90 of about 16 nM.

TABLE 5 NVP EFV Compound E-TMC278 wild type 81 1.4 0.4 100I 597 35 0.4101E 547 5 1.6 103N 2,879 28 0.3 106A 2,983 23 0.2 108I — 2 0.3 138K 641.3 0.4 179D 161 6 0.6 179E 158 5 0.4 181C 10,000 2 1.3 188C 3,764 5 0.1188H 241 9 0.2 188L 10,000 78 2.0 190A 4,101 8 0.3 190S 10,000 275 0.1225H 171 2 0.3 227C 1,816 36 2.0 227L 78 0.3 0.3 234I 45 NT 0.3 236L 411 0.3 100I + 103N 10,000 10,000 7.9 101E + 103N 7,033 84 0.5 103N + 181I10,000 37 1.0 227L + 106A 10,000 8 1.0

Development of Resistance In Vitro

NNRTIs are highly selective inhibitors of HIV-1 but their currentclinical use is limited by the rapid emergence of NNRTI (cross-)resistance. The rate of resistance emergence against compound E-TMC278and the first generation NNRTIs nevirapine and efavirenz was compared invitro.

MT4 cells were infected with wild type HIV-1 at high multiplicity ofinfection (>1 infectious virus per cell, to maximize the geneticdiversity of the virus population) in the presence of variousconcentrations of compound E-TMC278 (40, 200, 1000 and 5000×IC50), andwere monitored twice a week for virus replication. Emerging virus wascollected for pheno- and genotyping. Cultures without evidence of virusreplication were further sub-cultivated in the presence of the sameconcentration of inhibitor for a total duration of 30 days (10passages).

Resistance to nevirapine emerged within 3-6 days, at all testedconcentrations. Breakthrough virus harboured the typical Y181C mutation.The same experiments with efavirenz resulted in the selection of G190Eat all concentrations (up to 5 μM) within 3 to 7 days. Compound E-TMC278did not select for resistant virus within 30 days using wild-type virus.If a double resistant mutant K103N+Y181C (IC50 0.8 nM) was used insteadof wild type virus, resistance did emerge at all tested concentrations.Starting from the single mutants Y181C (IC50 1.3 nM) or 103N (IC50 0.3nM), virus breakthrough did not occur at 40 and 200 nM, but did occur at10 nM.

In this experimental setting of high genetic diversity, HIV-1, resistantto first generation NNRTIs, was selected very rapidly. Resistant virusesharboured only one mutation. In contrast, emergence of HIV-1, resistantto compound E-TMC278 was delayed or did not occur.

Cardiovascular and Pulmonary Safety of Compound E-TMC278

Compound E-TMC278 had little or no effect on cardiovascular andpulmonary parameters in vivo at plasma levels covering and exceeding thetargeted plasma levels in man and at concentrations in vitro covering orexceeding the anti-viral concentration in vitro.

Example 3: In Vitro Models to Test the Ability of Compound E-TMC278 toPrevent HIV Infection Via Sexual Intercourse or Related Intimate ContactBetween Partners

For instance, in one model, monocyte-derived dendritic cells (MO-DC)were infected for 2 hours with the monotropic HIV strain Ba-L at amultiplicity of infection (MOI) of 10⁻³. After infection, cells werewashed 6 times and resuspended in 10% BCS at 400.000 cells/ml.Autologous CD4(+) T cells were purified out of the lymphocyte fractionof the same elutration as the MO-DC and used at a concentration of 2×10⁶cells/ml ((ratio MO-DC/CD4(+) T:1/5).

A serial dilution of a compound of formula (I) (test compound) was addedto the MO-DC/CD4(+) T cell co-cultures. Each experiment was done in96-well plates, in which each cup contained 50 μl of MO-DC, 50 μl ofCD4(+) T cells and 100 μl of test compound. Half of the culture medium,with test compound, was refreshed twice weekly. Supernatants wereanalysed in ELISA after 14 days of culture. To determine antiviralactivity, the test compound concentration able to suppress 50% of theviral replication at the end of the primary cultures (EC50) wasmeasured. For compound E-TMC278, the EC50 value was 0.55 nM.

Example 4: Formulations

Tablet formulation of the following composition:

Emtricitabine 300 mg Tenofovir diisoproxyl fumarate 300 mg E-TMC278hydrochloride salt 110 mg HPMC 2910 15 mPa · s 24 mg Polysorbate 20 6 mgCrosspolyvidone 18 mg Lactose monohydrate 43 mg Magnesium stearate 3 mgTalcum 6 mg

The active ingredients and lactose are fluidised and sprayed with asolution of HPMC and polysorbate in water (at an equivalent of 120ml/tablet). Subsequently crosspolyvidone is added, while still beingfluidised, followed by magnesium stearate and talcum. The thus obtainedgranulate is compressed into 13 mm cylindrical tablets using standardcompressing equipment.

1. A combination comprising4-[[4-[[4-(2-cyanoethenyl)-2,6-dimethylphenyl]-amino]-2-pyrimidinyl]-amino]-benzonitrile,or a stereoisomeric form thereof; or a pharmaceutically acceptable saltthereof, lamivudine, and abacavir or a pharmaceutically acceptable saltthereof; wherein the4-[[4-[[4-(2-cyanoethenyl)-2,6-dimethylphenyl]-amino]-2-pyrimidinyl]-amino]-benzonitrile,or a stereoisomeric form thereof; or a pharmaceutically acceptable saltthereof; the lamivudine; and the abacavir or pharmaceutically acceptablesalt thereof are therapeutically effective HIV inhibitors at a dose thatcan be administered once daily.
 2. The combination of claim 1, whereinthe4-[[4-[[4-(2-cyanoethenyl)-2,6-dimethylphenyl]-amino]-2-pyrimidinyl]-amino]-benzonitrile,or a stereoisomeric form thereof; or a pharmaceutically acceptable saltthereof, isE-4-[[4-[[4-(2-cyanoethenyl)-2,6-dimethylphenyl]-amino]-2-pyrimidinyl]-amino]-benzonitrile.3. The method of claim 1, wherein the4-[[4-[[4-(2-cyanoethenyl)-2,6-dimethylphenyl]-amino]-2-pyrimidinyl]-amino]-benzonitrile,or a stereoisomeric form thereof; or a pharmaceutically acceptable saltthereof, isE-4-[[4-[[4-(2-cyanoethenyl)-2,6-dimethylphenyl]-amino]-2-pyrimidinyl]-amino]-benzonitrilehydrochloride.
 4. The combination of claim 1, comprising between 10 mgand 500 mg of the4-[[4-[[4-(2-cyanoethenyl)-2,6-dimethylphenyl]-amino]-2-pyrimidinyl]-amino]-benzonitrileor a stereoisomeric form thereof.
 5. The combination of claim 1,comprising between 10 mg and 300 mg of the4-[[4-[[4-(2-cyanoethenyl)-2,6-dimethylphenyl]-amino]-2-pyrimidinyl]-amino]-benzonitrileor a stereoisomeric form thereof.
 6. The combination of claim 1,comprising about 25 mg of the4-[[4-[[4-(2-cyanoethenyl)-2,6-dimethylphenyl]-amino]-2-pyrimidinyl]-amino]-benzonitrileor a stereoisomeric form thereof.
 7. The combination of claim 1,comprising about 150 mg of the lamivudine.
 8. The combination of claim1, comprising abacavir sulfate.
 9. The combination of claim 1,comprising about 300 mg of the abacavir, or a pharmaceuticallyacceptable salt thereof.
 10. The combination of claim 1, furthercomprising zidovudine.
 11. The combination of claim 10, wherein thecombination comprises about 300 mg of zidovucine.
 12. The combination ofclaim 1, as a unitary pharmaceutically acceptable formulation.
 13. Thecombination of claim 12, wherein the unitary pharmaceutically acceptableformulation is selected from the group consisting of tablets, capsules,parenteral compositions, injectable solutions, and injectablesuspensions.
 14. The combination of claim 12, wherein the unitarypharmaceutically acceptable formulation is a tablet.
 15. A method oftreating HIV infection in a patient comprising administering to thepatient the combination of claim 1 once daily.